WO2020025653A2 - Acide polysialique et ses dérivés, composition pharmaceutique et procédé de production d'acide polysialique - Google Patents

Acide polysialique et ses dérivés, composition pharmaceutique et procédé de production d'acide polysialique Download PDF

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WO2020025653A2
WO2020025653A2 PCT/EP2019/070576 EP2019070576W WO2020025653A2 WO 2020025653 A2 WO2020025653 A2 WO 2020025653A2 EP 2019070576 W EP2019070576 W EP 2019070576W WO 2020025653 A2 WO2020025653 A2 WO 2020025653A2
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polysialic acid
acid
acetylgroup
derivatives
monomer
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PCT/EP2019/070576
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WO2020025653A3 (fr
Inventor
Alexander DITYATEV
Hristo VARBANOV
Shaobo Jia
Rita Gerardy-Schahn
Evgeni Ponimaskin
Hauke THIESLER
Herbert Hildebrandt
Timm FIEBIG
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Medizinische Hochschule Hannover (Mhh)
Deutsches Zentrum Fuer Neurodegenerative Erkrankungen E.V. (Dzne)
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Priority to US17/263,595 priority Critical patent/US20210301038A1/en
Priority to EP19753269.0A priority patent/EP3829717A2/fr
Publication of WO2020025653A2 publication Critical patent/WO2020025653A2/fr
Publication of WO2020025653A3 publication Critical patent/WO2020025653A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention relates to a polysialic acid according to the general formula (1) as given as follows and derivatives thereof: 8)Neu5Ac) n with n being an integer in the range from 6 to 13, for use in the prevention or treatment of a neurological and neuropsychiatric disorder.
  • the present invention also relates to a pharmaceutical composition comprising as an active ingredient said polysialic acid and/or derivatives and/or pharmaceutically acceptable salts thereof.
  • the present invention relates to a method of producing said polysialic acid and/or derivatives and/or pharmaceutically acceptable salts thereof.
  • PolySia polysialic acid
  • NCAM neural cell adhesion molecule
  • AD Alzheimer's disease
  • polySia-NCAM expression in the entorhinal cortex is significantly decreased and negatively correlated with hyperphosphorylated Tau levels, one of the hallmarks of AD.
  • PolySia is a linear homopolymer that is composed of varying numbers of sialic acid residues (Hildebrandt et at., 2010; Schnaar et al., 2014). PolySia may though adopt a helical structure and can be identified with specific probes, such as antibodies and endo-A/- acylneuraminidase (Endo-N). In the adult brain, polySia is typically found at very low levels. However, it is found in distinct regions where neural plasticity, remodeling of neural connections or neurogenesis is ongoing, such as the hippocampus, subventricular zone (SVZ), thalamus, prefrontal cortex and amygdala. In the mature hippocampus, polySia-NCAM is involved in regulation of A/-methyl-D-aspartate receptor (NMDAR)-dependent synaptic plasticity.
  • NMDAR A/-methyl-D-aspartate receptor
  • the number of monomers in natural polysialic acids can reach 200.
  • Most of the polySia chains on the mammalian glycoprotein neural cell adhesion molecule (NCAM) consist of a variable degree of sialic acid monomers. Extended polysialic acid chains have been observed on glycoproteins of human neuroblastoma.
  • the objective of the present invention is to comply with this need.
  • polysialic acids and/or derivatives thereof and/or pharmaceutically acceptable salts thereof according to the present invention may compensate for polysialic acid deficiency and be used for therapeutic targeting of extrasynaptic NMDARs in a neurological and neuropsychiatric disorder, such as schizophrenia, a tauopathy, or amyloidosis.
  • a neurological and neuropsychiatric disorder such as schizophrenia, a tauopathy, or amyloidosis.
  • the efficiancy of polysialic acids according to the present invention was clearly demonstrated for improvements of cortical synaptic and cognitive functions.
  • the present invention deals with a polysialic acid according to the general formula (1) as given as follows and derivatives thereof:
  • Neu5Ac is N-acetylneuraminic acid
  • n is an integer in the range from 6 to 13
  • derivatives of the polysialic acid are substituted with at least one sugar, acetylgroup or acylgroup at at least one monomer of the polysialic acid,
  • the present invention may comprise the polysialic acid as mentioned above, wherein n is an integer in the range from 10 to 13.
  • the present invention may also envisage the polysialic acid as mentioned above, wherein the polysialic acid inhibits the activation of heterodimeric GluN1/GluN2B or heterotrimeric GluN1/GluN2A/GluN2B-containing NMDA receptors.
  • the present invention may encompass the polysialic acid as mentioned above, wherein said derivatives are substituted with at least one sugar, acetylgroup or acylgroup at one monomer of the polysialic acid.
  • the present invention comprises the polysialic acid as mentioned above, wherein said derivatives are substituted with at least one sugar, acetylgroup or acylgroup at the one monomer at the non-reducing end of the polysialic acid.
  • the present invention may further envisage the polysialic acid as mentioned above, wherein the at least one sugar is glucose, N-acetylglucosamine, N-acetylgalactosamine, galactose, fucose, mannose or xylose.
  • Also comprised by the present invention may be a polysialic acid as mentioned above, wherein said derivatives thereof have the formula (2) as given as follows:
  • Ri is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R 8 , Rio, R 12 , R 13 is independently from each other a hydrogen;
  • R 4 is a hydroxyl group or an O-acetylgroup;
  • R 6 is NHCOCH 3 ;
  • R 9 is a hydroxyl group or an O-acetylgroup
  • Rn is a hydroxyl group or a hydrogen
  • R 14 is a hydroxyl group or an O-acetylgroup.
  • R is a hydroxyl group
  • R 6 is NHCOCH 3
  • Rg is a hydroxyl group or an O-acetylgroup
  • R 14 is an O-acetylgroup.
  • R 6 is NHCOCH 3
  • R g is a hydroxyl group or an O-acetylgroup
  • R 1 is an O- acetylgroup
  • n of formula (2) is an integer in the range from 10 to 13.
  • the present invention may also encompass the polysialic acid as mentioned above, wherein the polysialic acid is chemically linked via its reducing end to a nano-carrier.
  • Also comprised by the present invention may be a polysialic acid as mentioned above, wherein the neurological and neuropsychiatric disorder is schizophrenia, tauopathy, bipolar disorder, depression, epilepsy, Huntington’s disease, multiple sclerosis, amyotrophic lateral sclerosis or stroke.
  • the neurological and neuropsychiatric disorder is schizophrenia, tauopathy, bipolar disorder, depression, epilepsy, Huntington’s disease, multiple sclerosis, amyotrophic lateral sclerosis or stroke.
  • tauopathy comprises Alzheimer’s disease, frontotemporal dementia (FTD), primary age-related tauopathy (PART), chronic traumatic encephalopathy, progressive supranuclear palsy (PSP), corticobasal degeneration, dementia with Lewy Bodies (DLB), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), argyrophilic grain disease (AGD), glial globular tauopathy, Pick’s diseases, and Parkinson’s disease.
  • FTD frontotemporal dementia
  • PART primary age-related tauopathy
  • PSP progressive supranuclear palsy
  • DLB dementia with Lewy Bodies
  • ATD argyrophilic grain disease
  • glial globular tauopathy Pick’s diseases, and Parkinson’s disease.
  • the present invention also comprises a pharmaceutical composition
  • a pharmaceutical composition comprising as an active ingredient the polysialic acid and/or derivatives and/or pharmaceutically acceptable salts thereof as mentioned above, optionally comprising a pharmaceutical acceptable carrier.
  • the present invention may also encompass the pharmaceutical composition as described above, wherein the pharmaceutically composition comprises a pharmaceutically acceptable carrier, wherein the pharmaceutical acceptable carrier is a solid pharmaceutical acceptable carrier, preferably lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate or stearic acid, a liquid pharmaceutical acceptable carrier, preferably sugar syrup, peanut oil, olive oil or water, or a gaseous pharmaceutical acceptable carrier, preferably carbon dioxide or nitrogen.
  • the pharmaceutical acceptable carrier is a solid pharmaceutical acceptable carrier, preferably lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate or stearic acid
  • a liquid pharmaceutical acceptable carrier preferably sugar syrup, peanut oil, olive oil or water
  • a gaseous pharmaceutical acceptable carrier preferably carbon dioxide or nitrogen.
  • compositions as described above, wherein the pharmaceutical composition is administered intranasal, oral, dermal, rectal, parenteral, preferably intranasal. Additionally, the present invention may also encompass the pharmaceutical composition as described above, wherein parenteral administration comprises intravitreal injection, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, perfusion, infusion or topical administration.
  • the present invention may envisage the pharmaceutical composition as described above for use in the prevention or treatment of a neurological and neuropsychiatric disorder. Additionally, the present invention may comprise the pharmaceutical composition as described above, wherein the neurological and neuropsychiatric disorder is schizophrenia, tauopathy, bipolar disorder, depression, epilepsy, Huntington’s disease, multiple sclerosis, amyotrophic lateral sclerosis or stroke.
  • the neurological and neuropsychiatric disorder is schizophrenia, tauopathy, bipolar disorder, depression, epilepsy, Huntington’s disease, multiple sclerosis, amyotrophic lateral sclerosis or stroke.
  • tauopathy comprises Alzheimer’s disease, frontotemporal dementia (FTD), primary age-related tauopathy (PART), chronic traumatic encephalopathy, progressive supranuclear palsy (PSP), corticobasal degeneration, dementia with Lewy Bodies (DLB), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), argyrophilic grain disease (AGD), glial globular tauopathy, Pick’s diseases, and Parkinson’s disease.
  • FTD frontotemporal dementia
  • PART primary age-related tauopathy
  • PSP progressive supranuclear palsy
  • corticobasal degeneration dementia with Lewy Bodies
  • FTDP-17 dementia with Lewy Bodies
  • ATD argyrophilic grain disease
  • glial globular tauopathy Pick’s diseases, and Parkinson’s disease.
  • the present invention comprises a method of producing the polysialic acid and/or derivatives and/or pharmaceutically acceptable salts thereof as mentioned above, comprising: a) dissolving colominic acid in acetic acid;
  • step b) stopping the reaction of step a) with NaOH;
  • Figure 1 shows the effects of polySia fragments on evoked NMDAR-EPSCs in endoNF- treated mPFC acute slices from C57BL/6J mice.
  • Figure 1 (A, C) shows a scheme of recordings (top left) and representative examples (top right) and time courses of normalized amplitudes (bottom) of evoked NMDAR-EPSCs (evoked by stimulation (S) at layers I I/I 11 and recorded (R) in layer V pyramidal neurons at the holding potential of -60 mV) during basal recording, followed by successive bath application of DP12 (NANA12) (1 pg/ml) ( Figure 1 (A)) or DP1 (NANA1 ) (10 pg/ml, control compound) ( Figure 1 (C)) and the GluN2B-subunit specific antagonist Ro 25-6981 (0.3 mM) in sham- and endoNF-treated slices.
  • Figure 1 (B) shows a bar graph summarizing mean levels of NMDAR-EPSC inhibition after DP12 and DP12 + Ro 25-6981 application (cells presented in Figure 1 (A)).
  • Figure 1 (C) shows no effect of DP1 , but a significant inhibition of NMDAR-EPSC amplitude by Ro 25-6981 (P ⁇ 0.05, paired Student’s t test) in endoNF- treated slices.
  • Figure 1 (D) shows a bar graph summarizing effects of DP1 (NANA1 ), DP5 (NANA5), and DP12 (NANA12) on NMDAR-EPSC amplitude in endoNF-incubated slices (normalized to the mean Ro 25-6981 -mediated inhibition of amplitude in endoNF slices).
  • One- way ANOVA revealed a significant difference between treatment groups (P ⁇ 0.001 ).
  • * P ⁇ 0.05, *** P ⁇ 0.001 Holm-Sidak post hoc test. Numbers of cells recorded and mice used are presented in bars. Data are shown as mean ⁇ SEM values.
  • Figure 2 shows the effects of the polySia mimetic tegaserod on synaptically evoked NMDAR-EPSCs in mPFC slices from C57BL/6 mice.
  • Figure 2(A) shows a scheme of recordings (top left) and representative examples (top right, averages of 8 - 10 traces) of evoked NMDAR-EPSCs before (black) and after the application of tegaserod (0.1 mM, grey) recorded from layer V pyramidal cells in sham- and endoNF-incubated slices.
  • NMDAR-EPSCs were recorded in whole-cell voltage-clamp mode at -60 mV and isolated pharmacologically in a conventional manner, but in the presence of the 5-HT 4 receptor antagonist RS 39604 (10 mM), to dissect the polySia-related effects of tegaserod.
  • Figure 3 shows the impact of endoNF, Ro 25-6981 , DP12, and GlyT1 inhibitors on long- term potentiation (LTP) in polySia-depleted mPFC slices.
  • Figure 3(A) shows the time courses of normalized mean slope of field excitatory postsynaptic potentials (fEPSPs), showing impaired theta-burst stimulation (TBS)-induced LTP in endoNF- compared with sham-treated slices.
  • Figure 3(B) shows normal LTP levels in endoNF-treated slices recorded in the presence of the GluN2B-selective antagonist Ro 25-6981 (0.3 pM, abbreviated as Ro25) or DP12 (1 pg/ml).
  • Figure 3(C) shows both glycine transporter type 1 inhibitors, sarcosine (0.75 mM) and SSR 504734 (3 pM), restored LTP magnitude in endoNF- treated slices to the level measured in sham-treated slices. Delivery of 5x theta-burst stimulation (TBS) is indicated by arrows. Insets show averages of 30 fEPSPs recorded during 10 min before TBS (black) and 50-60 min after TBS (gray), respectively, in each condition. The mean slope of fEPSPs recorded 10 min before TBS was taken as 100%.
  • Figure 3(D) shows a bar graph summarizing mean levels of LTP measured 50-60 min after TBS application in Figure 3(A-C).
  • Figure 4 shows the restoration of LTP in the mPFC of ST8SIA4-deficient mice by Ro 25- 6981 , DP12 and sarcosine.
  • Figure 4(A) shows reduced levels of TBS-induced LTP in slices from StSsia ⁇ mice compared with St8sia4 +I+ controls.
  • Figures 4(B-D) show fully restored LTP levels in slices from St8sia4 ⁇ / ⁇ mice and unchanged LTP levels in St8sia4 +I+ mice in the presence of Ro 25-6981 (0.3 pM) (Figure 4(B)), polySia DP12 (1 pg/ml) ( Figure 4(C)), or sarcosine (0.75 mM) ( Figure 4(D)).
  • Figure 4(E) shows the summary of mean LTP levels in St8sia4 mice in Figure 4(A-D).
  • Figure 5 shows novel object recognition and recency tests in St8sia4 ⁇ ⁇ mice and after in vivo intra-mPFC injection of endoNF in C57BL/6 mice.
  • Figure 5(A) shows the experimental design.
  • sarcosine 600 mg/kg b.w., intraperitoneally
  • DP12 / DP1 (1 mg/kg, intranasally
  • DMB-DP12 / DMB-DP2 (2 mg/kg, intranasally) were delivered 30 min before the encoding phase, and retention of memory was evaluated 2 hours later, in the retrieval phase.
  • reagents were applied 30 min before the first of two encoding phases separated by 1 hour interval, and recency memory was then assessed 10 min later.
  • Figure 5(B-D) upper panels show exploration times, while lower panels show discrimination ratio (%) in all tested groups and trials.
  • An arrow shows the timing of endoNF injection.
  • Intranasal delivery of DP12, but not DP1 , at day 5 could also restore cognitive function compared to a control compound ( * P ⁇ 0.05, ** P ⁇ 0.01 , *** P ⁇ 0.001 , paired t test).
  • Untreated Stesiaf ⁇ - mice (n 10) failed to discriminate between the most recent (R) and least recent (L) objects (D).
  • # P ⁇ 0.05, + P 0.0603, Holm-Sidak post hoc test. Data are shown as mean ⁇ SEM values.
  • Figure 6 shows two-photon in vivo imaging of DP12 penetration in the mPFC.
  • FIG. 6(A) shows EGFP signal that was used to do imaging in the same position before (baseline, 0 h) as well as 0.5 h, 3 h, and 24 h after intranasal administration of drugs (10 mg/ kg, 3 mice per group).
  • Figure 6(B) shows DMB signal in the same position where the EGFP imaging was done. Scale bar, 50 pm.
  • Figure 6(C) provides a statistical summary of imaging data from three mice. The DMB/EGFP ratio was used for quantification to compensate for unaccounted variability in laser intensity. Note the increase of fluorescent signal 0.5 - 3 hours after intranasal delivery of reagents. Data are shown as mean ⁇ SEM values.
  • Figure 7 shows that DP12 rescues mPFC LTP and recency memory in a mouse model of tauopathy overexpressing mutated human Tau[R406W] protein in the mPFC.
  • Figure 7(A) shows a scheme of experiment.
  • Figure 7(B) shows AAV-driven expression of GFP- Tau[R406W] and control GFP in the medial prefrontal cortex 1 month after injection of AAVs. Expression of GFP-Tau[R406W] leads to increased level of phosphorylated Tau (p-Tau). Scale bar, 100 pm.
  • Figure 7(C) shows that injection of Tau-GFP results in impaired mPFC LTP, which could be rescued by application of 0.3 pM DP12. Inserts above LTP profiles show fEPSPs recorded 10 min before and 50 - 60 min after induction of LTP. The bar plot depicts mean + SEM values of LTP 50 - 60 min after induction.
  • Figure 8 shows that application of DP12 increased LTP in CA3-CA1 synapses in 5xFAD mice.
  • Figure 8(A) shows profiles of LTP induced in 5xFAD mice by theta-burst stimulation (at time 0) in control and 1 pg/ml (approx. 0.3 mM) DP12 bath-perfused slices.
  • Figure 8(B) shows mean ⁇ SEM of LTP levels 50 - 60 min after theta-burst stimulation. **P ⁇ 0.01 , t-test. Numbers of slices recorded and mice used are presented in bars.
  • Figure 9 shows a column run after loading of 5 g hydrolysed colominic.
  • oligosialic acid with an n being 5 (DP5) and polysialic acid with an n being 26 (DP26) are indicated.
  • the Figure shows isolation of single DPs from a DNAPad OO column. The elution gradient is monitored by the conductivity of the elution buffer (see legend). The recording at 280 nm demonstrates that the sample was free of protein contaminants. The elution profile of polysialic acid recorded at 214 nm demonstrates base line separation of single DPs. The positions of DP5 and DP26 are indicated by black arrows.
  • Figure 10 shows that the oligosaccharide acceptor was successfully elongated with the supplied modified sialic acid.
  • the elution time of the unmodified acceptor DP9 is indicated by a vertical line.
  • Figure 11 shows the isolation and purification of 90Ac-modified oligosaccharides by FPLC-AEC.
  • the fractions containing DP9-90AC (F44-47), DP10-9OAC (F49-52) and DP11- 90Ac (F54-57) are indicated. These fraction were pooled to obtain avDP10-9OAc.
  • Figure 12(A) shows a modified scheme of recency test (increased difficulty) used in experiments with 5xFAD mice.
  • Figure 12(C) shows that intranasal administration of DP12 restored cognitive function in the recency test.
  • Figure 13 shows the effects of DP12, avDP1 Q-90Ac, and DP10 on neural cell viability measured using the MTT assay.
  • the cell viability represents means + SEMs of corrected optical densities in treated groups, which were expressed in % of mean value measured in control wells (treated with H 2 0 as vehicle) in the same plate.
  • DP10 had a significant potentiating effect (p ⁇ 0.001 , Kruskal-Wallis test, 11 cultures per group) and Dunnett’s post hoc test revealed a significant increase in cell viability in 30, 100 and 300 nM DP10-treated groups as compared to the vehicle group (p ⁇ 0.01 ).
  • the term "at least" preceding a series of elements is to be understood to refer to every element in the series.
  • the term“at least one” refers to one or more such as two, three, four, five, six, seven, eight, nine, ten and more.
  • the term “about” means plus or minus 10%, preferably plus or minus 5%, more preferably plus or minus 2%, most preferably plus or minus 1 %.
  • the present invention relates to a polysialic acid according to the general formula (1) as given as follows: (a(2 -> 8)Neu5Ac) n
  • Neu5Ac is N-acetylneuraminic acid
  • n is an integer in the range from 6 to 13
  • the present invention relates to a polysialic acid according to the general formula (1) as given as follows: (a(2 -> 8)Neu5Ac) n
  • Neu5Ac is N-acetylneuraminic acid
  • n is an integer in the range from 6 to 13
  • derivatives for use in the prevention or treatment of a neurological and neuropsychiatric disorder, wherein said derivatives are substituted with at least one sugar, acetylgroup, or acylgroup at at least one monomer of the polysialic acid, optionally linked to at least one nano-carrier, and pharmaceutically acceptable salts thereof.
  • polysialic acid refers to homopolymers of sialic acid comprising 6-13 monomers, preferably 10, 1 1 , 12 or 13 monomers. Additionally or alternatively, the term“polysialic acid” according to the present invention means sialic acid homopolymers with Neu5Ac residues at the position 5, wherein the homopolymers are linked via an a-2.8-linkage and which are recognized by the monoclonal antibody 735 (Frosch, M., Gorgen, I., Boulnois, G.J., Timmis, K.N., Bitter-Suermann, D. (1985). Proc. Natl. Acad. Sci.
  • oligosialic acid refers to homopolymers of sialic acid comprising 5 or less, e.g. 5, 4 or 3, monomers. The same may apply for the derivatives of the polysialic acid as defined herein and the respective pharmaceutically acceptable salts thereof.
  • the term“monomer” refers to a molecule being able to undergo polymerization, thereby contributing constitutional units to the essential structure of a macromolecule. Large numbers of monomers combine to form polymers in a process called polymerization. Polymers are large molecules (macromolecules) composed of many repeating units (many monomers), where the number of monomers is in priniciple infinite. However, oligomers only consist of a few monomer units.
  • the term“monomer” may preferably mean one sialic acid molecule of the whole polysialic acid molecule with an integer n as described herein. The same may apply for the derivatives of the polysialic acid as defined herein and the respective pharmaceutically acceptable salts thereof.
  • “DPn” refers to the polysialic acid as defined and used in the present invention with the respective n being an integer as described herein.
  • the same may apply for the derivatives of the polysialic acid as defined herein and the respective pharmaceutically acceptable salts thereof.
  • Polysialic acid is an extended homopolymer of sialic acid.
  • Sialic acid is a N- or O- substituted derivative of neuraminic acid.
  • Polysialic acid/ polySia (PSA) is found on glycoproteins, and is a component of the capsular polysaccharides of certain pathogenic bacteria. In bacteria, the sialic acid monomers of PSA can be linked by a 2.8 or a 2.9 linkage to form polysialic acid. In humans, the sialic acid monomer of PSA is linked by a 2.8 linkage and is acetylated at position 5.
  • the sialic acid monomer N-acetylated at position 5 usually is abbreviated Neu5Ac (N- acetylneuraminic acid).
  • the monosaccharide Neu5Ac is denoted 5-acetamido-2,4-dihydroxy-6- (1 ,2,3-trihydroxypropyl)oxane-2-carboxylic acid according to the IUPAC nomenclature.
  • the Neu5Ac monomers are linked by a (2 - 8) linkage to form polysialic acid molecules in the present invention. This may be achieved by using glycosidic bonds between the Neu5Ac monomer units of the polysialic acid.
  • the a(2 - 8) linkages result in a highly flexible linear molecule, while at low pH the chemical structure of the polymer forms lactones, resulting in a more rigidified structure.
  • the number of monomers in polysialic acid can reach 200.
  • Most of the PSA chains on the mammalian glycoprotein neural cell adhesion molecule (NCAM) consist of a variable degree of sialic acid monomers. Extended polysialic acid chains have been observed on glycoproteins of human neuroblastoma.
  • NCAM neural cell adhesion molecule
  • the neural cell adhesion molecule is a transmembrane glycoprotein that promotes cell-cell and cell-extracellular matrix adhesion.
  • NCAM regulates multiple processes, such as neurite outgrowth, neuronal migration, and synaptogenesis.
  • the removal of polySia is performed by bacterial endosialidases, such as endoNF.
  • polySia-NCAM is involved in /V-methyl-D-aspartate receptor (NMDAR)-dependent synaptic plasticity.
  • a NMDA receptor is a glutamate receptor and ion channel protein found in the membrans of nerve cells. It forms glutamate-gated ion channels that have central roles in neuronal communication and plasticity throughout the brain. It is activated when glutamate and glycine (or D-serine) bind to it, and when activated it allows positively charged ions, most importantly Ca 2+ , to flow through the cell membrane.
  • Dysfunctions of NMDARs are involved in several central nervous system disorders, including stroke, chronic pain, dementia and schizophrenia.
  • One hallmark of NMDARs is that their activity can be allosterically regulated by a variety of extracellular small ligands.
  • the receptor is a heteromeric complex that interacts with multiple intracellular proteins by three different subunits: GluN 1 , GluN2 and GluN3.
  • GluN1 has eight different isoforms (or also called slicing variants) generated by alternative splicing from a single gene.
  • NMDARs form heterotetrameric complexes usually consisting of two GluN 1 and two GluN2 subunits.
  • polysialic acid inhibits GluN2B-containing NMDARs only at the low micromolar glutamate concentrations characteristic of the extrasynaptic space and because most extrasynaptic NMDARs are thought to be GluN2B heterodimers, the inventors found it plausible that polysialic acid specifically inhibits extrasynaptic NMDARs such as extrasynaptic GluN 1/2B receptors or extrasynaptic GluN 1/GluN2A/GluN2B receptors.
  • polysialic acid according to the general formula (1 ) as described elsewhere herein indeed inhibits the activation of heterodimeric GluN 1/GluN2B and may inhibit heterotrimeric GluN 1/GluN2A/GluN2B-containing NMDARs.
  • polysialic acid according to the general formula (1 ) may be used for therapeutic targeting of extrasynaptic NMDARs in a neurological and neuropsychiatric disorder, further being used in the prevention or treatment of a neurological and neuropsychiatric disorder.
  • a neurological and neuropsychiatric disorder is schizophrenia, tauopathy, bipolar disorder, depression, epilepsy, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis or stroke.
  • a neurological and neuropsychiatric disorder is schizophrenia, tauopathy, or amyloidosis.
  • a neurological and neuropsychiatric disorder is schizophrenia or tauopathy.
  • a neurological and neuropsychiatric disorder is a tauopathy.
  • tauopathy comprises Alzheimer’s disease, frontotemporal dementia (FTD), primary age-related tauopathy (PART), chronic traumatic encephalopathy, progressive supranuclear palsy (PSP), corticobasal degeneration, dementia with Lewy Bodies (DLB), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), argyrophilic grain disease (AGD), glial globular tauopathy, Pick’s diseases, Parkinson’s disease.
  • a tauopathy is Alzheimer’s disease.
  • the present invention comprises the polysialic acid according to the general formula (1 ) as defined elsewhere herein and pharmaceutical acceptable salts thereof for use in the prevention or treatment of schizophrenia or a tauopathy.
  • the present invention comprises the polysialic acid according to the general formula (1 ) as defined elsewhere herein and pharmaceutical acceptable salts thereof for use in the prevention or treatment of a tauopathy, preferably of Alzheimer’s disease.
  • Physiologically acceptable salts of the compounds of the present invention are in particular salts with a non-toxic salt component and preferably are pharmaceutically utilizable salts. They can contain inorganic or organic salt components.
  • Such salts can be formed, for example, from compounds of the present invention, which contain an acidic group, for example a carboxylic acid group (HO-CO-) or a sulfonic acid group (HO-S(0) 2 -) and non-toxic, inorganic or organic bases.
  • Suitable bases are, for example, alkali metal compounds or alkaline earth metal compounds, such as sodium hydroxide, potassium hydroxide, sodium carbonate or sodium hydrogencarbonate, or ammonia, organic amino compounds and quaternary ammonium hydroxides. Reactions of compounds of the present invention with bases for the preparation of the salts are in general carried out according to customary procedures in a solvent or diluent.
  • advantageous salts of acidic groups are in many cases sodium, potassium, magnesium or calcium salts or ammonium salts, which can also carry one or more organic groups on the nitrogen atom.
  • Compounds of the present invention which contain a basic, i.e.
  • protonatable, group for example an amino group or another basic heterocycle
  • group for example an amino group or another basic heterocycle
  • physiologically acceptable acids for example, a salt with hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, acetic acid, benzoic acid, methanesulfonic acid, p-toluenesulfonic acid, which in general can be prepared from the compounds of the present invention by reaction with an acid in a solvent or diluent according to customary procedures.
  • the ratio of the salt components can deviate upward or downward from the stoichiometric ratio, such as the molar ratio 1 : 1 or 1 :2 in the case of the acid addition salt of a compound of the present invention containing one or two basic groups with a monovalent acid, and vary depending on the applied conditions.
  • the present invention comprises also salts containing the components in a non-stoichiometric ratio, and an indication that an acid addition salt of a compound of the present invention contains an acid in equimolar amount, for example, also allows for a lower or higher amount of acid in the obtained salt, for example, about 0.8 or about 1 .1 mol of acid per mol of compound of the present invention.
  • the compounds of the present invention simultaneously contain an acidic and a basic group in the molecule, the invention also includes internal salts (betaines, zwitterions) in addition to the salt forms mentioned.
  • the term “pharmaceutically acceptable” may in particular mean approved by a regulatory agency or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.
  • the“n” of formula (1 ) is an integer in the range from 10 to 13, such as 10, 11 , 12, or 13. Having said that, the present invention comprises a polysialic acid having the following formula: (a(2 -> 8)Neu5Ac) 10 , (a(2 -> 8)Neu5Ac)n ,
  • the polysialic acid according to the general formula (1 ) may optionally be linked to at least one nano-carrier.
  • said polysialic acid of the present invention may be linked to at least one nano-carrier, preferably one nano-carrier.
  • the term“nano-carrier” refers to nanomaterial being used as a transport module for another substance (e.g. for the polysialic acid of the present invention).
  • Commonly used nano-carriers may include, but are not limited to, lipid-based carriers, such as micelles and liposomes, polymers, carbon-based materials, polymeric nanoparticles, dendrimers, carbon nanotubes, and gold nanoparticles and other substances.
  • Nano-carriers range from sizes of diameter 1 - 1000 nm. In the present invention a nano-carrier of ⁇ 200 nm is preferably used. Nano-carriers are useful in the drug delivery process. They are able to deliver drugs to site-specific targets, allowing drugs to be delivered in certain organs or cells but not in others. Thus, this site-specificity is a major therapeutic benefit since it prevents drugs from being delivered to the wrong places.
  • the nano- carrier being used in the present invention thus helps to deliver polysialic acid according to the general formula (1 ) as described elsewhere to its target/ acting place (e.g. the brain), where it is able to inhibit extrasynaptic NMDARs in a neurological and neuropsychiatric disorder.
  • the polysialic acid according to the general formula (1 ) may be chemically linked via its reducing end to a nano-carrier.
  • the term“chemically linked” refers to a direct glycosidic linkage formed between the anomeric OH- group at position 2 of Neu5Ac and a second substrate or the use of a bifunctional linker that on one hand side reacts with the anomeric OH in position 2 of Neu5Ac and on the other hand reacts with a chemical group (usually a free amino-group) in the second substrate.
  • the term“reducing end of the polysialic acid” in this context and as used throughout the present invention refers to the Neu5Ac-residue of said polysialic acid according to the general formula (1 ), in which the anomeric center is not involved into the formation of an a-2,8- glycosidic linkage, even if it is conjugated through the hydroxyl-group at C2 (position 2).
  • Biological polysialic acid is usually part of a glycan forming a posttranslational modification on proteins (primarily membrane anchored proteins). The conjugation to the glycan core structure (N-glycan or O-glycan) is through the reducing end.
  • cell-type-specific polysialic acid membrane anchors such as a membrane anchored protein molecule (e.g., N -glycosylated membrane anchored protein, O -glycosylated membrane anchored protein or a glycosylated, glycosylphosphoinositol (GPI)-membrane anchored protein). It may also be possible to link a nano-carrier to said reducing end of the polysialic acid according to the general formula (1 ) as indicated above.
  • the term “non-reducing end of polysialic acid” in this context and as used through the present invention refers to the terminal sugar being a-2,8-glycosidically linked to the previous according to the general formula (1 ).
  • prevention refers to a complete inhibition of the development of a neurological and neuropsychiatric disorder in a subject by applying the polysialic acid according to the general formula (1 ) as defined elsewhere herein and/or derivatives thereof and/or pharmaceutically acceptable salts thereof or a pharmaceutical composition comprising as an active ingredient the polysialic acid and/or derivatives and/or pharmaceutically acceptable salts thereof as described elsewhere herein.
  • treatment refers to halting the progression of a neurological and neuropsychiatric disorder in a subject, which has already been suffering from a neurological and neuropsychiatric disorder when the treatment with the polysialic acid according to the general formula (1 ) as defined elsewhere herein and/or derivatives thereof and/or pharmaceutically acceptable salts thereof or a pharmaceutical composition comprising as an active ingredient the polysialic acid and/or derivatives and/or pharmaceutically acceptable salts thereof as described elsewhere herein has been initiated.
  • a derivative in the context of the present invention refers to the polysialic acid according to the general formula (1 ) as described elsewhere herein being further substituted with at least one sugar, acetylgroup or acylgroup at at least one monomer of the polysialic acid.
  • the term“substituted at at least one monomer” means that a group (e.g. a hydrogen or hydroxylgroup or the like) or more groups at (a) certain position(s) (e.g. at C1 and/or C2 and/or C3 and/or C4 and/or C5 and/or C6 and/or C7 and/or C8 and/or C9 of said polysialic acid) at at least one monomer of said polysialic acid of the present invention is being substituted/ replaced with at least one sugar, acetylgroup or acylgroup, whereby said sugar, acetylgroup, or acylgroup replaces the group (e.g.
  • a hydrogen or hydroxyl group or the like being attached to said C-molecule (e.g. a hydrogen on C3 for example) or whereby said sugar, acetylgroup or acylgroup replaces more groups being attached at several, different C-molecules at at least one monomer, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen monomers of the polysialic acid of the present invention.
  • said derivative of the present invention may be substituted with at least one sugar, acetylgroup or acylgroup at only one monomer of said polysialic acid according to the general formula (1). It is especially preferred, that said derivative of the polysialic acid according to formula (1) may be substituted with at least one sugar, acetylgroup or acylgroup at the one monomer at the non-reducing end of the polysialic acid.
  • substituted at one monomer means that a group (e.g. a hydrogen or hydroxylgroup or the like) or more groups at (a) certain position(s) (e.g. at C1 and/or C2 and/or C3 and/or C4 and/or C5 and/or C6 and/or C7 and/or C8 and/or C9 of said polysialic acid) at one monomer of said polysialic acid of the present invention is being substituted/ replaced with at least one sugar, acetylgroup or acylgroup, whereby said sugar, acetylgroup or acylgroup replaces the group (e.g.
  • a hydrogen or hydroxyl group or the like being attached to said C-molecule (e.g. a hydrogen on C3 for example) or whereby said sugar, acetylgroup or acylgroup replaces more groups being attached at several, different C-molecules at one monomer of the polysialic acid of the present invention.
  • said derivative of the present invention may be substituted with at least one sugar, acetylgroup or acylgroup at the one monomer at the non-reducing end of said polysialic acid according to the general formula (1 ).
  • the term“substituted at the one monomer at the non-reducing end” means that a group (e.g. a hydrogen or hydroxylgroup or the like) or more groups at (a) certain position(s) (e.g.
  • non-reducing end of polysialic acid refers to the terminal sugar a-2,8-glycosidically linked to the previous according to the general formula (1).
  • Polysialic acid chain growth occurs by the addition of sialic acid monomers to the non-reducing terminus of the growing chain. This may also be called tail growth.
  • capping in the context of the present invention means herein that polysialic acid according to the present invention is substituted only at its non-reducing end sugar. It is especially preferred that such a capping is an O-acetylation.
  • said sugar molecule may be linked to said specific position at at least one monomer, at one monomer or at the one monomer at the non-reducing end of said polysialic acid of the present invention according to the general formula (1) via glycosidic linkage.
  • Said linkage is a type of covalent bond that joins a carbohydrate (sugar) molecule to another group (e.g. the polysialic acid of the present invention), which may or may not be another carbohydrate.
  • Glycosidic bonds are known to the person skilled in the art.
  • sugar according to the present invention is to be understood as meaning monosaccharides and disaccharides, which commonly are referred to as sugars.
  • the at least one sugar may be glucose, N-acetylglucosamine, N-acetylgalactosamine, galactose, fucose, mannose or xylose.
  • glucose, N-acetylglucosamine, N-acetylgalactosamine, galactose, fucose, mannose and xylose are essential sugars within the human body.
  • the polysialic acid can comprise one terminal sugar molecule or be glycosidically bound to two or more sugars. Polysialic acid glycosidically linked to one or more sugar molecules can result in improved pharmacokinetics.
  • the sugar molecule may be linked to the reducing end of said polysialic acid chain being linked via an a-(2,3)-linkage.
  • glycosidically bound is to be understood as meaning the polysialic acid that is bound to a further saccharide molecule, or other molecules capable of forming a glycosidic bond such as amino acids.
  • polysialic acid of the present invention may be substituted with at least one glucose, at least one N- acetylglucosamine, at least one N-acetylgalactosamine, at least one galactose, at least one fucose, at least one mannose or at least one xylose at at least one monomer, at one monomer or at the one monomer at the non-reducing end of said polysialic acid of the present invention according to the general formula (1 ).
  • said derivatives of said polysialic acid according to the general formula (1 ) are substituted with at least one acetylgroup at at least one monomer, at one monomer or at the one monomer at the non-reducing end of said polysialic acid of the present invention according to the general formula (1 ). More preferably, said derivatives of said polysialic acid according to the general formula (1 ) are substituted with at least one O-acetylgroup at at least one monomer, at one monomer or at the one monomer at the non-reducing end of said polysialic acid of the present invention according to the general formula (1 ).
  • said derivatives of said polysialic acid according to the general formula (1 ) are substituted with at least one O- acetylgroup at the one monomer at the non-reducing end of said polysialic acid of the present invention according to the general formula (1 ).
  • the O-acetylation(s) of said derivative(s) of the present invention may be at position 4 (C4) and/or position 7 (C7) and/or position 9 (C9) at the one monomer at the non-reducing end of said polysialic acid according to the general formula (1 ).
  • the O-acetylation(s) of said derivative(s) of the present invention may be at position 7 (C7) and/or position 9 (C9) at the one monomer at the non-reducing end of the polysialic acid according to the general formula (1 ) as described herein.
  • the O-acetylation of said derivative(s) of the present invention may be at position 9 (C9) at the one monomer at the non-reducing end of the polysialic acid according to the general formula (1 ) as described herein.
  • derivatives of said polysialic acid according to the general formula (1) being O-acetylated at position 9 at the one monomer at the non-reducing end and with a length of n being 10 refer to a polysialic acid according to the general formula (1), wherein n is 9, and an additional sialic acid according to the formula (a(2 -> 8)Neu5Ac) being additionally O-acetylated at position 9.
  • the sialic acid according to the formula (a(2 8)Neu5Ac) being additionally O-acetylated at position 9 is at the non-reducing end of said polysialic acid according to the general formula (1 ), so that the complete polysialic acid has in total an n being 10.
  • derivatives of said polysialic acid according to the general formula (1) being O-acetylated at position 9 at the one monomer at the non-reducing end of the polysialic acid with a length of n being 11 refer to a polysialic acid according to the general formula (1), wherein n is 10, and an additional sialic acid according to the formula (a(2 -> 8)Neu5Ac) being additionally O-acetylated at position 9.
  • the sialic acid according to the formula (a(2 -> 8)Neu5Ac) being additionally O-acetylated at position 9 is at the non-reducing end of said polysialic acid according to the general formula (1 ), so that the complete polysialic acid has in total an n being 11.
  • derivatives of said polysialic acid according to the general formula (1) being O-acetylated at position 9 at the one monomer at the non-reducing end of the polysialic acid with a length of n being 12, refer to a polysialic acid according to the general formula (1), wherein n is 11 and an additional sialic acid according to the formula (a(2 - 8)Neu5Ac) being additionally O-acetylated at position 9.
  • the sialic acid according to the formula (a(2 - 8)Neu5Ac) being additionally O-acetylated at position 9 is at the non-reducing end of said polysialic acid according to the general formula (1 ), so that the complete polysialic acid has in total an n being 12.
  • derivatives of said polysialic acid according to the general formula (1) being O-acetylated at position 9 at the one monomer at the non-reducing end of the polysialic acid with a length of n being 13, refer to a polysialic acid according to the general formula (1), wherein n is 12 and an additional sialic acid according to the formula (a(2 8)Neu5Ac) being additionally O-acetylated at position 9.
  • the sialic acid according to the formula (a(2 8)Neu5Ac) being additionally O-acetylated at position 9 is at the non-reducing end of said polysialic acid according to the general formula (1 ), so that the complete polysialic acid has in total an n being 13.
  • n is an integer in the range from 10 to 13, such as 10, 11 , 12 or 13, thereby producing the“derivatives” of the present invention, may be carried out with CMP activated 9-O-acetylated sialic acid and polysialic acid according to the general formula (1), wherein n is an integer in the range from 9 to 12, preferably in the molecular ratio 1.5 : 1 using the engineered bacterial derived NmB-polyST clone F116 (Keys et ai, 2014).
  • CMP-9-O-acetylated sialic acid was synthesized from 0.5 mM 5-acetamido-9-0- acetyl-3,5-dideoxy-D-gylcero-galacto-non-2-ulosonic acid and 0.5 mM CTP using 0.5 mM of the CMP-sialic acid synthase (CMAS) from Neisseria meningitidis serogroup B. Further, 9-0- acetylated sialic acid was then transferred from CMP-9-O-acetylated sialic acid onto 0.1 mM of the acceptor oligosaccharide (e.g.
  • CMAS CMP-sialic acid synthase
  • n is an integer of 9 using 0.5 mM of the distributive Neisseria meningitidis serogroup B polysialyltransferase (clone F116 (1)) as mentioned elsewhere herein.
  • the reaction buffer being used in the method described above concerning the synthesis of said derivatives of the present invention may comprise sodium phosphate, MgCI 2 and glycerol.
  • the reaction buffer may comprise 50 mM phosphate, 10 mM MgCI 2 and 5% glycerol and a pH 8.0. Reactions may be incubated at 25°C. After 1 h (HPLC; DNAPac PA 100 column; Dionex, Sunnyvale, USA), modified polymer may be separated from non-modified material by preparative HPLC as it is described in paragraphs [00160] - [00161]
  • Said derivative(s) of the present invention may have the formula (2) as given as follows:
  • Ri is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R 8 , Ri 0 , R I2 , R 13 is independently from each other a hydrogen; iii) R 4 is a hydroxyl group or an O-acetylgroup;
  • R 6 is NHCOCH 3 ;
  • R 9 is a hydroxyl group or an O-acetylgroup
  • Rn is a hydroxyl group or hydrogen
  • R 14 is a hydroxyl group or an O-acetylgroup.
  • Said derivative(s) of the present invention may also have the formula (2) as given as follows:
  • R-i is a carboxyl group
  • each of R 2 , R 3 , R 5 R 7 R 8 R 10 , R 12 , R 13 is independently from each other a hydrogen; iii) R 4 is a hydroxyl group or an O-acetylgroup;
  • R 6 is NHCOCH 3 ;
  • R 9 is a hydroxyl group or an O-acetylgroup
  • Rn is a hydroxyl group
  • R 14 is a hydroxyl group or an O-acetylgroup.
  • Said derivative(s) of the present invention may also have the formula (2) as given as follows:
  • R-i is a carboxyl group
  • R 13 is independently from each other a hydrogen; iii) R is a hydroxyl group or an O-acetylgroup; iv) R 6 is NHCOCH 3 ;
  • R 9 is a hydroxyl group or an O-acetylgroup
  • Rn is hydrogen
  • R 14 is a hydroxyl group or an O-acetylgroup.
  • n of formula (2) as described above is an integer in the range from 10 to 13, such as 10, 11 , 12 or 13.
  • the present invention may comprise (a) derivative(s) according to formula (2) of the polysialic acid of the present invention, wherein at the one monomer at the non- reducing end:
  • R is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R B , Rio, R12, R13 is independently from each other a hydrogen;
  • R 4 is a hydroxyl group or an O-acetylgroup;
  • R 6 is NHCOCH 3 ;
  • R 9 is a hydroxyl group or an O-acetylgroup
  • Rn is a hydroxyl group or hydrogen
  • R 14 is a hydroxyl group or an O-acetylgroup
  • n is an interger in the range from 10 to 13, such as 10, 11 , 12 or 13,
  • the present invention may also comprise (a) derivative(s) according to formula (2) of the polysialic acid of the present invention, wherein at the one monomer at the non- reducing end:
  • R-i is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R 8 , R 10 , R12, R13 is independently from each other a hydrogen;
  • R 4 is a hydroxyl group or an O-acetylgroup;
  • R 6 is NHCOCH 3 ;
  • Rg is a hydroxyl group or an O-acetylgroup
  • Rn is a hydroxyl group
  • R 14 is a hydroxyl group or an O-acetylgroup
  • n is an interger in the range from 10 to 13, such as 10, 11 , 12 or 13,
  • the present invention may also comprise (a) derivative(s) according to formula (2) of the polysialic acid of the present invention, wherein at the one monomer at the non- reducing end:
  • R1 is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R e , R10, R12, R13 is independently from each other a hydrogen;
  • R 4 is a hydroxyl group or an O-acetylgroup;
  • R 6 is NHCOCH 3 ;
  • R 9 is a hydroxyl group or an O-acetylgroup
  • Rn is hydrogen
  • R 14 is a hydroxyl group or an O-acetylgroup
  • n is an interger in the range from 10 to 13, such as 10, 11 , 12 or 13,
  • the present invention may also comprise (a) derivative(s) having the formula (2) as described above, wherein at the one monomer at the non-reducing end of polysialic acid:
  • R is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R 8 , Rio, R12, R13 is independently from each other a hydrogen; iii) R 4 is an O-acetylgroup;
  • R 6 is NHCOCH 3 ;
  • Rg is a hydroxyl group
  • Rn is a hydroxyl group or a hydrogen
  • R 14 is a hydroxyl group
  • the present invention may also comprise (a) derivative(s) having the formula (2) as described above, wherein at the one monomer at the non-reducing end of polysialic acid:
  • R-i is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R 8 , R10, R12, R13 is independently from each other a hydrogen; iii) R 4 is an O-acetylgroup;
  • R 6 is NHCOCH 3 ;
  • Rg is a hydroxyl group
  • Rn is a hydroxyl group
  • R 14 is a hydroxyl group
  • the present invention may also comprise (a) derivative(s) having the formula (2) as described above, wherein at the one monomer at the non-reducing end of polysialic acid:
  • R1 is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R 8 , R 10 , R12, R13 is independently from each other a hydrogen; iii) R 4 is an O-acetylgroup;
  • R 6 is NHCOCH 3 ;
  • R 9 is a hydroxyl group
  • Rn is a hydrogen
  • R 14 is a hydroxyl group
  • Also comprised by the present invention may be (a) derivative(s) having the formula (2) as described above, wherein at the one monomer at the non-reducing end of polysialic acid:
  • R is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R 8 , Rio, R12, R13 is independently from each other a hydrogen; iii) R 4 is an O-acetylgroup;
  • R 6 is NHCOCH 3 ;
  • R 9 is a hydroxyl group
  • Rn is a hydroxyl group or a hydrogen
  • R 14 is a hydroxyl group
  • n is an integer in the range from 10 to 13, such as 10, 11 , 12 or 13,
  • [00112] Also comprised by the present invention may be (a) derivative(s) having the formula (2) as described above, wherein at the one monomer at the non-reducing end of polysialic acid:
  • R-i is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R 8 , R10, R12, R13 is independently from each other a hydrogen; iii) R 4 is an O-acetylgroup;
  • R 6 is NHCOCH 3 ;
  • Rg is a hydroxyl group
  • Rn is a hydroxyl group
  • R 14 is a hydroxyl group
  • n is an integer in the range from 10 to 13, such as 10, 11 , 12 or 13,
  • Also comprised by the present invention may be (a) derivative(s) having the formula (2) as described above, wherein at the one monomer at the non-reducing end of polysialic acid:
  • R1 is a carboxyl group; ii) each of R 2 , R 3 , R 5 , R 7 , R 8 , R 10 , R12 ⁇ R13 is independently from each other a hydrogen; iii) R 4 is an O-acetylgroup;
  • R 6 is NHCOCH 3 ;
  • R 9 is a hydroxyl group
  • Rn is a hydrogen
  • R 14 is a hydroxyl group
  • n is an integer in the range from 10 to 13, such as 10, 11 , 12 or 13,
  • the present invention may also comprise (a) derivative(s) having the formula (2) as described above, wherein at the one monomer at the non-reducing end:
  • R is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R 8 , R10, R12, R13 is independently from each other a hydrogen; iii) R 4 is a hydroxyl group;
  • R 6 is NHCOCH 3 ;
  • Rg is an O-acetylgroup
  • Rn is a hydroxyl group or a hydrogen
  • R 1 is an O-acetylgroup
  • the present invention may also comprise (a) derivative(s) having the formula (2) as described above, wherein at the one monomer at the non-reducing end:
  • R-i is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R 8 , Ri 0 , R12, R13 is independently from each other a hydrogen; iii) R 4 is a hydroxyl group;
  • R 6 is NHCOCH 3 ;
  • Rg is an O-acetylgroup
  • Rn is a hydroxyl group
  • R 14 is an O-acetylgroup
  • the present invention may also comprise (a) derivative(s) having the formula (2) as described above, wherein at the one monomer at the non-reducing end:
  • R1 is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R 8 , R 10 , R12 ⁇ R13 is independently from each other a hydrogen
  • iii) R 4 is a hydroxyl group
  • R 6 is NHCOCH 3 ;
  • R 9 is an O-acetylgroup
  • Rn is a hydrogen
  • R 14 is an O-acetylgroup
  • Also comprised by the present invention may be (a) derivative(s) having the formula (2) as described above, wherein at the one monomer at the non-reducing end:
  • R is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R e , R10, R12, R13 is independently from each other a hydrogen; iii) R is a hydroxyl group;
  • R 6 is NHCOCH 3 ;
  • R 9 is an O-acetylgroup
  • Rn is a hydroxyl group or a hydrogen
  • R 14 is an O-acetylgroup
  • n is an integer in the range from 10 to 13, such as 10, 11 , 12 or 13,
  • Also comprised by the present invention may be (a) derivative(s) having the formula (2) as described above, wherein at the one monomer at the non-reducing end:
  • R-i is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R 8 , R 10 , RI 2 , R13 is independently from each other a hydrogen; iii) R 4 is a hydroxyl group;
  • R 6 is NHCOCH 3 ;
  • Rg is an O-acetylgroup
  • Rn is a hydroxyl group
  • R 14 is an O-acetylgroup
  • n is an integer in the range from 10 to 13, such as 10, 11 , 12 or 13,
  • Also comprised by the present invention may be (a) derivative(s) having the formula (2) as described above, wherein at the one monomer at the non-reducing end:
  • R1 is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R 8 , R 10 , RI 2 , R13 is independently from each other a hydrogen; iii) R is a hydroxyl group; iv) R 6 is NHCOCH 3 ;
  • R 9 is an O-acetylgroup
  • Rn is a hydrogen
  • R 14 is an O-acetylgroup
  • n is an integer in the range from 10 to 13, such as 10, 11 , 12 or 13,
  • the present invention comprises (a) derivative(s) having the formula (2) as described above, wherein at the one monomer at the non-reducing end:
  • R is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R 8 , Rio, R12, R13 is independently from each other a hydrogen;
  • R 4 is a hydroxyl group;
  • R 6 is NHCOCH 3 ;
  • R 9 is a hydroxyl group
  • Rn is a hydroxyl group or a hydrogen
  • R 14 is an O-acetylgroup
  • the present invention comprises (a) derivative(s) having the formula (2) as described above, wherein at the one monomer at the non-reducing end:
  • R-i is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R 8 , R 10 , RI 2 , RI 3 is independently from each other a hydrogen; iii) R 4 is a hydroxyl group;
  • R 6 is NHCOCH 3 ;
  • Rg is a hydroxyl group
  • Rn is a hydroxyl group
  • R 14 is an O-acetylgroup
  • the present invention comprises (a) derivative(s) having the formula (2) as described above, wherein at the one monomer at the non-reducing end:
  • R1 is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R 8 , R10, R12, R13 is independently from each other a hydrogen; iii) R is a hydroxyl group; iv) R 6 is NHCOCH 3 ;
  • R 9 is a hydroxyl group
  • Rn is a hydrogen
  • R 14 is an O-acetylgroup
  • the present invention comprises (a) derivative(s) of the present invention having the formula (2) as described above, wherein at the one monomer at the non-reducing end:
  • R is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R B , Rio, R12, R13 is independently from each other a hydrogen;
  • R 4 is a hydroxyl group;
  • R 6 is NHCOCH 3 ;
  • R 9 is a hydroxyl group
  • Rn is a hydroxyl group or a hydrogen
  • R 14 is an O-acetylgroup
  • n is an integer in the range from 10 to 13, such as 10, 11 , 12 or 13,
  • the present invention comprises (a) derivative(s) of the present invention having the formula (2) as described above, wherein at the one monomer at the non-reducing end:
  • R-i is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R 8 , R10, R12, R13 is independently from each other a hydrogen; iii) R 4 is a hydroxyl group;
  • R 6 is NHCOCH 3 ;
  • Rg is a hydroxyl group
  • Rn is a hydroxyl group
  • R 14 is an O-acetylgroup
  • n is an integer in the range from 10 to 13, such as 10, 11 , 12 or 13,
  • the present invention comprises (a) derivative(s) of the present invention having the formula (2) as described above, wherein at the one monomer at the non-reducing end:
  • R1 is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R e , R10, R12, R13 is independently from each other a hydrogen; iii) R 4 is a hydroxyl group;
  • R 6 is NHCOCH 3 ;
  • R 9 is a hydroxyl group
  • Rn is a hydrogen
  • R 14 is an O-acetylgroup
  • n is an integer in the range from 10 to 13, such as 10, 11 , 12 or 13,
  • the present invention comprises (a) derivative(s) having the formula (2) as described above, wherein at the one monomer at the non-reducing end:
  • R is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R 8 , Rio, R 12 , R 13 is independently from each other a hydrogen; iii) R is an O-acetylgroup;
  • R 6 is NHCOCH 3 ;
  • R 9 is an O-acetylgroup
  • Rn is a hydroxyl group or hydrogen
  • R 14 is an O-acetylgroup
  • the present invention comprises (a) derivative(s) having the formula (2) as described above, wherein at the one monomer at the non-reducing end:
  • R-i is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R 8 , R 10 , R 12 , R 13 is independently from each other a hydrogen; iii) R 4 is an O-acetylgroup;
  • R 6 is NHCOCH 3 ;
  • Rg is an O-acetylgroup
  • Rn is a hydroxyl group
  • R 14 is an O-acetylgroup
  • the present invention comprises (a) derivative(s) having the formula (2) as described above, wherein at the one monomer at the non-reducing end:
  • R 1 is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R 8 , R 10 , R 12 , R 13 is independently from each other a hydrogen; iii) R 4 is an O-acetylgroup;
  • R 6 is NHCOCH 3 ;
  • R 9 is an O-acetylgroup
  • Rn is a hydrogen
  • R 14 is an O-acetylgroup
  • the present invention comprises (a) derivative(s) of the present invention having the formula (2) as described above, wherein at the one monomer at the non-reducing end:
  • R is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R e , Rio, R12, R13 is independently from each other a hydrogen; iii) R is an O-acetylgroup;
  • R 6 is NHCOCH 3 ;
  • R 9 is an O-acetylgroup
  • Rn is a hydroxyl group or a hydrogen
  • R 14 is an O-acetylgroup
  • n is an integer in the range from 10 to 13, such as 10, 11 , 12 or 13,
  • the present invention comprises (a) derivative(s) of the present invention having the formula (2) as described above, wherein at the one monomer at the non-reducing end:
  • R-i is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R 8 , R10, R12, R13 is independently from each other a hydrogen; iii) R 4 is an O-acetylgroup;
  • R 6 is NHCOCH 3 ;
  • Rg is an O-acetylgroup
  • Rn is a hydroxyl group
  • R 14 is an O-acetylgroup
  • n is an integer in the range from 10 to 13, such as 10, 11 , 12 or 13,
  • the present invention comprises (a) derivative(s) of the present invention having the formula (2) as described above, wherein at the one monomer at the non-reducing end:
  • R1 is a carboxyl group; ii) each of R 2 , R 3 , R 5 , R 7 , R 8 , R 10 , R12 ⁇ R13 is independently from each other a hydrogen; iii) R 4 is an O-acetylgroup;
  • R 6 is NHCOCH 3 ;
  • R 9 is an O-acetylgroup
  • Rn is a hydrogen
  • R 14 is an O-acetylgroup
  • n is an integer in the range from 10 to 13, such as 10, 11 , 12 or 13,
  • derivatives of the present invention may also be optionally linked to a least one nano-carrier as it has already been described for the polysialic acid of the present invention.
  • paragraphs [0072] to [0074] may be applicable to said derivative as described above as well.
  • a cell free production of polysialic acid according to the present invention is also possible and may use a Maltose-Binding-Protein(MBP)-tagged engineered bacterial enzyme (MBP-NmBpolyST-F116) as described in Keys et a ⁇ . (2014).
  • the protein may be solid phase fixed to an amylose matrix (GE Healthcare; no. 28918779) and 30 min incubated at 25°C with CMP-Neu5Ac and a DP3 primer in 50 mM Tris-HCI, pH 8.0, 25 mM KCI, 20 mM MgCI 2 , 5 % glycerol.
  • the size of products can be simply controlled by the ratio DP3:CMP-Sia (e.g. the production of DP12 would request the ratio 1 :9). Reactions may be carried out in 1 - 2 ml volumes and may be stopped by centrifugation. Supernatants may be further processed as described in Figure 9.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising as an active ingredient the polysialic acid and/or derivatives and/or pharmaceutically acceptable salts thereof as defined elsewhere herein.
  • said pharmaceutical composition may comprise as an active ingredient the polysialic acid and/or derivatives and/or pharmaceutically acceptable salts thereof as defined elsewhere herein and a pharmaceutically acceptable carrier.
  • “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the "pharmaceutically acceptable carrier” may be in the form of a solid, semisolid, liquid, gaseous or combinations thereof.
  • the carrier is suitable for intranasal, enteral (such as oral), dermal, rectal, topical or parenteral administration (such as intravenous, intramuscular, subcutaneous, spinal or epidermal administration (e.g., by injection or infusion)).
  • the pharmaceutically acceptable carrier is a solid pharmaceutical acceptable carrier, preferably lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate or stearic acid.
  • the pharmaceutically acceptable carrier is a liquid pharmaceutical acceptable carrier, preferably sugar syrup, peanut oil, olive oil or water.
  • the pharmaceutically acceptable carrier is a gaseous pharmaceutical acceptable carrier, preferably carbon dioxide or nitrogen.
  • the polysialic acid according to the general formula (1 ) as described in present invention and/or derivatives thereof and/or pharmaceutical acceptable salts thereof are preferably administered to a patient in need thereof via a pharmaceutical composition.
  • the pharmaceutical composition comprises the polysialic acid according to the general formula (1 ) as described in the present invention and/or derivatives thereof and/or pharmaceutical acceptable salts thereof and one or more pharmaceutically acceptable excipient(s).
  • excipient when used herein, is intended to indicate all substances in a pharmaceutical composition, which are not active ingredients (e.g., which are therapeutically inactive ingredients that do not exhibit any therapeutic effect in the amount/ concentration used), such as, e.g., carriers, binders, lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffers, flavoring agents, colorants, or antioxidants.
  • active ingredients e.g., which are therapeutically inactive ingredients that do not exhibit any therapeutic effect in the amount/ concentration used
  • carriers binders, lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffers, flavoring agents, colorants, or antioxidants.
  • the pharmaceutical composition of the present invention may comprise the polysialic acid according to the general formula (1 ) as described in the present invention and/or derivatives thereof and/or pharmaceutical acceptable salts thereof, one or more pharmaceutically acceptable excipient(s) and a pharmaceutically acceptable carrier.
  • compositions may contain salts, buffers, preserving agents, carriers and optionally other therapeutic agents.
  • the pharmaceutical composition may be administered to an individual intranasal, oral, dermal, rectal, topical or parenteral.
  • parenteral administration and “administered parenterally” as used herein mean modes of administration other than enteral administration (examples of enteral administration include oral and rectal administration), usually by injection, perfusion or infusion or topical application, and include, without limitation, intravitreal injection, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, as well as topical administration (e.g., epicutaneous or through mucous membranes (such as buccal, sublingual or vaginal)).
  • enteral administration include oral and rectal administration
  • topical administration e.g., epicutaneous or through mucous membranes (such as buccal, sublingual or vaginal)
  • an intranasally administration of the pharmaceutical composition comprising as an active ingredient the polysialic acid according to the general formula (1 ) as described in the present invention and/or derivatives and/or pharmaceutically acceptable salts thereof as defined elsewhere herein is preferred.
  • the pharmaceutical composition may also comprise adjuvants such as preservatives, wetting agents, emulsifying agents, pH buffering agents, and dispersing agents. Prevention of the presence of microorganisms may be ensured by sterilization procedures and/or by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents, which delay absorption such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents, pH buffering agents, and dispersing agents. Prevention of the presence of microorganisms may be ensured by sterilization procedures and/or by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid
  • the active compounds (the polysialic acid and/or derivatives and/or pharmaceutically acceptable salts thereof as defined elsewhere herein), which may be used in a suitable hydrated form, and/or the pharmaceutical compositions used according to the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art (cf., e.g., Remington, "The Science and Practice of Pharmacy” edited by Allen, Loyd V., Jr., 22 nd edition, Pharmaceutical Sciences, September 2012; Ansel et al., "Pharmaceutical Dosage Forms and Drug Delivery Systems", 7 th edition, Lippincott Williams & Wilkins Publishers, 1999).
  • a pharmaceutical composition can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
  • the pharmaceutical compositions containing one or more active compounds can be prepared with carriers that will protect the active compounds against rapid release, such as a controlled release formulation, including implants, transdermal patches and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. Methods for the preparation of such compositions are generally known to those skilled in the art. (See, e.g. Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.)
  • compositions typically are sterile and stable under the conditions of manufacture and storage.
  • Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the individuals to be treated. Each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the amount of active ingredient in particular, the amount of the polysialic acid according to the general formula (1) or according to any of the general formula (2) as described in the present invention and/or derivatives thereof and/or pharmaceutical acceptable salts thereof used according to the present invention, will range from about 0.01% to about 99%, preferably from about 0.1 % to about 70%, most preferably from about 1% to about 30%, wherein the reminder is preferably composed of the one or more pharmaceutically acceptable excipients.
  • the amount of active ingredient e.g., the polysialic acid according to the general formula (1) or according to the general formula (2) as described in the present invention and/or derivatives thereof and/or pharmaceutical acceptable salts thereof used according to the present invention, in a unit dosage form and/or when administered to an individual or used in therapy, may range from about 0.1 mg to about 10000 mg (for example, from about 1 mg to about 5000 mg, such as from about 10 mg to about 2000 mg) per unit, administration or therapy.
  • a suitable amount of such active ingredient may be calculated using the mass or body surface area of the individual, including amounts of between about 1 mg/kg and 500 mg/kg (for example between about 2 mg/kg and 250 mg/kg, such as between about 10 mg/kg and 100 mg/kg), or between about 1 mg/m 2 and about 4000 mg/m 2 (such as between about 10 mg/m 2 and about 3000 mg/m 2 or between about 100 mg/m 2 and about 2000 mg/m 2 ).
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions used according to the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start with doses of the compounds used according to the present invention at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of a composition used according to the present invention will be that amount of the compound, which is the lowest dose effective to produce a therapeutic effect.
  • Such an effective dose will generally depend upon the factors described above. It is preferred that administration be oral, intravenous, intramuscular, intraperitoneal or subcutaneous, preferably administered proximal to the site of the target.
  • the effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub- doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound used according to the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation/ composition.
  • the pharmaceutical composition used according to the invention can also, if desired, be presented in a pack, or dispenser device, which can contain one or more unit dosage forms containing the active compound.
  • the pack can for example comprise metal or plastic foil, such as blister pack.
  • the pack or dispenser device can be accompanied with instruction for administration.
  • the pharmaceutical acceptable composition comprising the polysialic acid according to the general formula (1 ) or according to the general formula (2) as defined elsewhere herein and/or derivatives thereof and/or pharmaceutical acceptable salts thereof for use in the prevention or treatment of a neurological and neuropsychiatric disorder.
  • the present invention comprises the pharmaceutical acceptable composition comprising the polysialic acid according to the general formula (1 ) or according to the general formula (2) as defined elsewhere herein and/or derivatives thereof and/or pharmaceutical acceptable salts thereof for use in the prevention or treatment of schizophrenia or tauopathy.
  • the present invention comprises the pharmaceutical acceptable composition comprising the polysialic acid according to the general formula (1 ) or according to the general formula (2) as defined elsewhere herein and/or derivatives thereof and/or pharmaceutical acceptable salts thereof for use in the prevention or treatment of a tauopathy, preferably of Alzheimer’s disease.
  • the present invention also comprises a method of producing the polysialic acid and/or derivatives and/or pharmaceutically acceptable salts thereof as described elsewhere herein, comprising a) dissolving colominic acid in acetic acid; b) stopping the reaction of step a) with NaOH, c) storing the mixture of step b); d) separating oligo- and polymers by chromatography with a NaCI gradient; e) obtaining the polysialic acid and/or derivatives and/or pharmaceutically acceptable salts thereof.
  • the term “manufacturing/ manufacture” may be used interchangeably with the term “producing/ produce” as used herein.
  • the term “producing the polysialic acid and/or pharmaceutically acceptable salts thereof” as described herein means that the polysialic acid and/or pharmaceutically acceptable salts thereof may be manufactured by using the method of the present invention.
  • the term“dissolving” means subjecting colominic acid to acidic hydrolysis in acetic acid (HAc).
  • acetic acid Preferably, at least 50 mg, 60 mg, 70 mg, 80 mg, 90 mg colominic acid is used for step a) of the method of the present invention.
  • 100 mg colominic acid is subjected to acidic hydrolysis in acetic acid (HAc) for step a) of the method of the present invention.
  • 100 mg colominic acid is subjected to acidic hydrolysis in 100 mM acetic acid (HAc) for 10 min at 70°C for step a) of the method of the present invention.
  • Those conditions had been demonstrated to generate DP5 - DP15 at high concentration.
  • the term“to stop/ stopping the reaction” in step b) of the method of the present invention refers to halt a certain reaction, in this case, the reaction of acidic hydrolysis of step a), by adjusting the pH to a certain value, which is alkaline.
  • the pH was adjusted to 10.5 by addition of NaOH. Even more preferably, 1 M NaOH was used to stop the reaction in step b) in the method of the present invention.
  • step b) of the method of the present invention which comprises the reacting components of step a) and NaOH was then stored at 4°C for 48 h to minimize intramolecular lactonization.
  • the term“to store/ storing” means transferring (e.g. filling) said mixture of step b) in a tube, container vessel, or dish and then warehousing said tube, container, vessel or dish being filled with said mixture of step b) of the method of the present invention into a refrigerator for a certain amount of time.
  • the term“to separate/ separating” as used herein refers to subjecting the mixture from step b) of the method of the present invention to exchange chromatography thereby separating oligosialic acids and polysialic acids of individual degrees of polymerization (DP).
  • the mixture was subjected to anion exchange chromatography applying a DNAPad OO (22 x 250 mm column; ThermoScientific), preceded by a guard column (4 x 50 mm; ThermoScientific).
  • said polysialic acid and/or pharmaceutically acceptable salts thereof as described elsewhere herein may also be formulated.
  • the polysialic acid and/or pharmaceutically acceptable salts thereof can be brought under conditions, where it is more stable.
  • buffer substances and additives such as sucrose, mild detergents, stabilizer and the like, known in the art can be used.
  • 5xFAD mice (Oakley et al., 2006) were bred at the animal facility of DZNE Magdeburg. St8siaf mice were backcrossed with C57BL/6J mice for > 8 generations. 5xFAD mice were backcrossed with C57BL/6J mice for > 10 generations. Mice heterozygous for St8sia4 were crossed to produce homozygous mutants and littermate wild-type controls.
  • mice For electrophysiological experiments in brain slices, we used adult 2- to 4-month- old C57BL/6J and Sf8s/a4 ⁇ _ mice and their respective age-matched wild-type littermates from both sexes. For behavioral experiments, we used male 2- to 3-month-old C57BL/6J mice, 7- to 10-month-old St8siaf ⁇ ⁇ mice and their wild-type littermates, 12- to 14-month-old male 5xFAD mice for the comparison with age- and gender-matched wildtype controls and for evaluation of DP12 and avDP10-9OAc, and 10- to 16-month-old 5xFAD mice of both genders for evaluation of DP10 and DP20. All mice were kept in a reverse light-dark cycle (12:12 hours, light on at 9:00 pm) with food and water ad libitum, and they were tested during the dark phase of the cycle, when mice are active.
  • Each mouse was quickly sacrificed by cervical dislocation. After decapitation, the brain was rapidly removed from the skull and placed in ice-cold artificial cerebrospinal fluid (aCSF) equilibrated with 95% 0 2 / 5% C0 2 , which was composed of the following (in mM): 250 sucrose, 24 NaHC0 3 , 25 glucose, 2.5 KCI, 1.25 NaH 2 P0 4 , 2 CaCI 2 , and 1.5 MgCI 2 (osmolarity 345 - 350 mOsm/kg, pH 7.4).
  • aCSF cerebrospinal fluid
  • coronal slices containing the prelimbic and infralimbic cortices were cut 350 pm or 400 pm thick, for patch clamp and extracellular recordings, respectively, in the same sucrose-containing aCSF. Then, the slices were transferred to a slice holding chamber filled with recording aCSF (osmolarity 300 - 305 mOsm/kg, pH 7.4) that contained 120 mM NaCI instead of sucrose (Eckhardt et al., 2000) and was continuously gassed with 95% 0 2 / 5% C0 2 .
  • aCSF osmolarity 300 - 305 mOsm/kg, pH 7.4
  • layer V pyramidal neurons were visualized using a SliceScope Pro 6000 equipped with a 40x water-immersion objective and infrared differential interference contrast microscopy (Scientifica, Uckfield, UK). Pyramidal cells were identified by their triangular soma and long apical dendrites, and synaptically evoked excitatory postsynaptic currents (EPSCs) were recorded using patch pipettes (3 - 5 MO) fabricated using borosilicate glass capillaries (wall thickness 0.315, length 100, outer diameter 1.5 mm, Hilgenberg) and a DMZ-Zeitz puller (Zeitz Instruments GmbH, Martinsried, Germany).
  • EPCs synaptically evoked excitatory postsynaptic currents
  • the intracellular pipette solution contained the following (in mM): 140.7 Cs-methane-sulfonate, 5 NaCI, 1 MgCI 2 , 0.2 EGTA, 10 HEPES, 3 ATP-Mg, 0.3 Na-GTP, and 3.1 QX-314 (osmolarity 295 mOsm/kg, adjusted to pH 7.2 with CsOH).
  • EPSCs were evoked by electrical pulses (0.2 ms) at 0.033 Hz via a glass pipette placed in layer ll/lll of the mPFC (identically to fEPSPs).
  • the slow NMDAR-mediated component of EPSCs was pharmacologically isolated at -60 mV in modified aCSF containing low Mg 2+ (0.1 mM) and high Ca 2+ (3.2 mM, for adjustment of divalent cation concentrations), the selective antagonist of a- amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors NBQX (10 mM), and the selective antagonist of g-aminobutyric acid (GABA) B receptors CGP-55845 (2 mM) (Chen et al., 2003).
  • the washout of Mg 2+ required at least 25 min.
  • Prefrontal slices 400 pm were transferred to a submerged-type recording MC membrane chamber (volume 2 ml; Scientific Systems Design Inc., Mississauga, Ontario, Canada) perfused with oxygenated recording ACSF at a flow rate of 4 ml/min and maintained at room temperature (22 - 25°C), unless stated otherwise in the“Results”-section given below.
  • Field EPSPs were evoked and recorded using thin-walled glass electrodes (borosilicate glass, wall thickness 0.188, length 100, and outer diameter 1.5 mm, Hilgenberg, Malsfeld, Germany) filled with recording aCSF in 400 pm-thick mPFC slices.
  • the stimulation electrode (0.3 - 0.5 MQ) was inserted into layers I I/Ill of the prelimbic cortex.
  • the recording electrode (2 - 2.5 MW) was placed in the vicinity of dendrites and cell bodies of pyramidal neurons in layer V (Huang et at., 2004).
  • basal synaptic transmission was recorded in each slice and the relationship between stimulus intensity and fEPSP slope was measured.
  • Basal fEPSPs were evoked at 0.05 Hz for at least 10 min.
  • the supramaximal slope was determined by gradually increasing the stimulation intensity until a population spike in the fEPSP became first visible. The stimulation intensity was adjusted to elicit a basal fEPSP with a slope of approximately 50% of the supramaximal value.
  • LTP was induced by application of five trains of theta-burst stimulation (TBS) with an inter-theta-train interval of 20 s (Brennaman ef a/., 2011 ). Each train consisted of 8 bursts delivered at 5 Hz. Each burst consisted of four pulses delivered at 100 Hz. Duration of pulses was 0.2 ms.
  • TBS ta-burst stimulation
  • the mean fEPSP slope was measured between 50 - 60 min after TBS delivery, and it was then normalized to mean baseline fEPSP slope during 0 - 10 min before TBS. Stimulus artifacts in representative fEPSP examples in figures were blanked to facilitate the perception of fEPSPs recorded in the mPFC. Recording and analysis of LTP in SteSia ⁇ mice was performed in a blind manner.
  • endosialidase NF endoNF
  • DP10 and DP12 short form of polysialic acid composed of 10 or twelve sialic acid residues
  • Sialic acid N-acetylneuraminic acid, DP1
  • DP5 pentamer of sialic acid sodium salt
  • DMB-labelled DP2 and DP12, and non-labelled avDP10-9OAc were synthesized in the laboratory of Rita Gerardy-Schahn (see sections Synthesis of DMB-DP2 and DMB-DP12 and Synthesis of avDP10-9OAc, respectively).
  • the GluN2B-selective antagonist (aR, S)-a-(4-hydroxyphenyl) ⁇ -methyl-4-(phenylmethyl)-1-piperidinepropanol maleate (Ro 25- 6981 maleate, abbreviated as Ro25), the subtype-unselective antagonist of NMDA receptors D- (-)-2-amino-5-phosphonopentanoic acid (AP5), the selective AMPA receptor antagonist 2,3- dioxo-6-nitro-1 ,2,3,4-tetrahydrobenzo[/]quinoxaline-7-sulfonamide disodium salt (NBQX), the antagonist of GABA A and glycine receptors picrotoxin, the GABA B receptor antagonist (2S)-3- [[(1 S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid hydrochloride (CGP-55845), and the selective 5-HT
  • the selective antagonist of the glial glycine transporter 1 (GlyT 1 ) N-methylglycine (sarcosine) and the partial agonist at the glycine modulatory site of NMDA receptors (R)-4-Amino-3- isoxazolidone, 4-Amino-3-isoxazolidinone (D-cycloserine, DCS) were purchased from Sigma Aldrich (Taufmün, Germany).
  • the selective antagonist of GlyT1 (2-chloro-N-[(S)-phenyl[(2S)- piperidin-2-yl]methyl]-3-trifluoromethyl benzamide (SSR 504734) was purchased from Axon Medchem (Groningen, Netherlands).
  • the 5-HT 4 receptor agonist (polySia mimetic) tegaserod maleate was purchased from Sequoia Research Products LTD (Pangbourne, UK). All antagonists and agonists were applied via the slice perfusion system. Sarcosine (0.75 mM) solution was freshly prepared prior to experiments in aCSF. For all other drugs, appropriate stock solutions in double-distilled water or DMSO were prepared according to the manufacturer’s recommendations and stored at -20°C.
  • the membrane-impermeable lidocaine and the antagonist of voltage-activated Na + channels A/-(2,6-Dimethylphenylcarbamoylmethyl)- triethylammonium chloride (QX-314 chloride) were purchased from Tocris Bioscience.
  • the maximal peak amplitude and decay time were analyzed using the PatchMaster software.
  • the decay time of EPSC was defined as the time interval beginning at 80% of the EPSC maximal amplitude (after the EPSC peak) and ending at 20% of that amplitude.
  • the NMDAR/AMPAR ratio in each cell was determined by dividing the average peak amplitude of the pharmacologically isolated NMDAR current by the average peak amplitude of AMPAR-mediated current recorded in normal aCSF. EPSC averages were based on 8 - 10 consecutive traces in each condition. For tonic currents, the NMDAR-mediated holding current was measured during 5 consecutive 2 s intervals before and 3 min after AP5 application.
  • the shift in holding current was assessed as the difference between mean amplitude values before and after the NMDAR antagonist application for each cell.
  • cortical fEPSPs were low-pass filtered at 500 Hz, and the rising slope of the fEPSP was measured during its initial linear phase using PatchMaster. Recordings of fEPSPs in which the fiber volley amplitude changed by more than 15% were excluded from the analysis.
  • CMP-90Ac-Sia was synthesized from 0.5 mM 5-acetamido-9-0-acetyl-3,5-dideoxy-D- gylcero-galacto-non-2-ulosonic acid (90Ac-Sia, kindly provided by Prof.
  • reaction was performed in a total volume of 2000 pi of reaction buffer containing 50 mM Tris pH 8.0, 25 mM KCI, 20 mM MgCI 2 , 5% glycerol and 0.001 U/pl pyrophosphatase.
  • reaction products were analyzed by HPLC-anion exchange chromatography (AEC) on a Prominence UFLC-XR (Shimadzu) equipped with a CarboPac PA-100 column (2 x 250 mm, Dionex). Samples were separated as described by Keys et ai, 2012, with the minor adjustment that H 2 0 and 1 M NaCI were used as mobile phases M1 and M2, respectively. 50 pi of sample were loaded for the detection of polysaccharide at 214 nm. Products were separated using an elution gradient consisting of a -2 curved gradient from 0 to 30% M2 over 4 min followed by a linear gradient from 30 to 84% M2 over 33 min.
  • AEC HPLC-anion exchange chromatography
  • Preparative AEC was performed on an AKTA pure 25 (GE Healthcare) equipped with a DNAPac PA100 22 x 250 mm including a 22 x 50 guard column at a flow rate of 4.0 ml/min. 10 mM Tris pH 8.0 and 10 mM Tris pH 8.0 + 1 M NaCI were used as mobile phases M1 and M2, respectively. Samples were separated using a combination of ten consecutive linear gradients:
  • Fraction collection was initiated in parallel with gradient iv.
  • the fraction size was set to 2 ml.
  • Fractions containing DPs of a specific size (DPs of known size had been used to equilibrate the column) were pooled as shown in Figure 11 and each DP was desalted in six consecutive steps of a decimal series of dilution using Amicon centrifugal devices with 3 kDa MWCO.
  • avDP10-9OAc fractions containing DP11 , DP10 and DP9 were pooled (see F49- 52 in Figure 11) and freeze-dried.
  • DP fragments of specific sizes were purified by anion exchange chromatography as described (Keys et ai, 2014).
  • the preparation of fluorescently labelled DPs followed the procedure described in (Keys et al., 2012) with some modifications.
  • coli K1 capsule polysaccharide exhibiting an average size of 40 sialic acid units were dissolved in 20 mM DMB (1 ,2-diamino-4,5- methylenedioxybenzene 2 HCI) with 1 M b-mercaptoethanol and 40 mM sodium dithionite to give a final concentration of 10 mg/ml.
  • the solution was mixed with an equal volume of ice cold 40 mM trifluoric acid (TFA) and incubated for 48 h at 4°C. Then the reaction was stopped by addition of NaOH until the pH of the mixture was 10.0. To revert lactonisation of polySia (induced by TFA treatment), the mixture was kept at room temperature for 24 h.
  • TFA trifluoric acid
  • DMB-labelled fragments were subsequently eluted using the following gradient: 0 to 8 % M2 over 12 ml, 8 to 20 % M2 over 56 ml, 20 to 45 % M2 over 208 ml with a fraction size of 4 ml.
  • the fractions containing DMP-DP12 were pooled and desalted using a desalting column.
  • DMB-DP2 was purified using a DNA Pac PA 100 column (Thermo Fisher Scientific, 22 x 250 mm), with water (M3) and 4 M ammonium acetate (M4) as liquid phases.
  • Acute injection of endoNF in vivo was done under a short isoflurane anesthesia.
  • a digitally controlled infusion system UltraMicroPump, UMP3, and Micro4 Controller, WPI, USA
  • the thin (135 pm) part of the needle went into the mouse brain during injection, whereas its thick part (430 pm) fit perfectly in the guide cannula (inside 483 pm).
  • Injections were performed as follows: 1 ) a mouse was anesthetized with 1 - 3 % isoflurane as during implantation; 2) it was put into the stereotaxic frame; 3) the dummy cannula was gently removed from the left guide cannula; 4) a NanoFil injecting needle was carefully placed inside of the guide cannula and advanced further down until reaching the marker (made with a permanent pen at the needle shank (430 pm) to visualize the point when the needle should reach the surface of the brain coming out of the guide cannula (equal to the length of guide cannula); 5) once the marker was reached, a slow (10 pm increment) step dial was used to deepen the needle into the brain; 6) endoNF (2 pg/pl) or vehicle was injected with the following settings: 250 nl per site as deep as 1.5 and 2.25 mm from the brain surface (total injection volume per hemisphere 500 nl, injection rate of 3 nl/s); 7) after injection was complete ( ⁇
  • mice were returned to their animal facility. Behavior experiments started on the day after injection at approximately the same time, so that we could perform cognitive tests 24 hours after injection.
  • AAVs adeno-associated viruses
  • AAVs were produced using the AAV helper free packaging systems AAV-DJ (Cell Biolabs, Inc.).
  • AAV-DJ Cell Biolabs, Inc.
  • HEK293 cells were transfected with the pAAV-DJ and pHelper constructs along with the viral expression constructs (pAAV vectors) encoding either eGFP or eGFP- Tau[R406W] under the control of a synapsin promotor. After three days, viruses were harvested using freeze-thaw cycles.
  • non-viral DNA is digested with Benzonase, and lysate is filtered through a 0.22 pm filter, washed with PBS and concentrated using Amicon Ultra-15 Centrifugal Filter Units with a cut off of 50 kDa. Titers were determined to be > 10 10 GC/ml using quantitative Real-Time PCR with primers for the WPRE element in the viral genome.
  • mice were anesthetized in a closed chamber using isoflurane (1 ml for 30 s). The mice were then mounted in a stereotaxic frame (Narishige, Tokyo, Japan) and chronically anesthetized using 1.5% isoflurane in combination with 0 2 .
  • viral vectors (AAV-eGFP and AAV-Tau-eGFP) were bilaterally injected (500 nl per site) in the prelimbic cortex (AP +1.7 mm; ML ⁇ 0.30 mm; DV -2.2 mm; relative to Bregma, according to the mouse brain atlas (Franklin and Paxinos, 2007) at an injection rate of 3 nl/s followed by an additional 5 min to allow diffusion.
  • the mice remained in their home cage for 4 weeks until the start of LTP recordings or recency test.
  • a white open field arena (50 x 50 x 30 cm) was used in the novel object recognition task. All behavior was video recorded and analyzed automatically by software (ANY- maze, version 4.99, Stoelting Co., Wood Dale, IL). The test was performed using a standard protocol (Leger et ai, 2013) that includes two phases: a) a familiarization/encoding phase: mice were placed for 10 min in the arena, during which they have to explore two identical objects positioned in the center of the arena; b) a test/ retrieval phase: one familiar object and one novel object were placed in the center of the arena, and mice were allowed to explore for 10 min. In the same trial, objects were counterbalanced, and between trials, different sets of objects were used. The interval between the encoding and retrieval phases was 2 h.
  • the recency test comprised of two encoding phases followed by a retrieval phase (modified from Nelson et ai, 2011 ).
  • the interval between the encoding phases was 1 h, and the interval between the second encoding phase and the retrieval phase was 10 min, except for experiments in 5xFAD mice when both intervals were 90 min to make the task more difficult.
  • animals were placed into the open field arena and allowed to explore a pair of identical objects for 10 min. Then, a different pair of identical objects was presented in the second encoding phase (10 min total exploration time).
  • the retrieval phase two different objects, one object from each encoding phase, were placed into the apparatus and animals were given 10 min to explore them.
  • Novelty detection was evaluated by calculating the discrimination ratio as follows: [novel object time - familiar object time] / [novel object time + familiar object time] x 100%. In the recency test, the discrimination ratio was calculated as follows: [least recent object time - most recent object time] / [least object time + most recent object time] x 100%. Unreliable measurements (about 10%), when animals spent in total ⁇ 10 s near to both objects, were excluded from analysis.
  • Results are expressed as the mean ⁇ SEM. SigmaPlot 12 or 13 and GraphPad 7 (La Jolla, CA, USA) software were used for data analysis and presentation. Differences between the two genotypes undergoing two different treatments were assessed by two-way repetitive measures ANOVA followed by the Holm-Sidak post hoc test. Differences between times spent exploring familiar and novel objects were assessed using a paired Student's t test for each genotype in each treatment. For all comparisons, values of p ⁇ 0.05 were considered significant. The Grubbs test was used to detect outliers (about 2%), which were excluded from analysis.
  • cryo-protective solution 25% glycerin (Carl Roth, Düsseldorf, Germany), 25% ethylene glycol (Carl Roth) in 0.24 M PB) and stained“free-floating” using immunohistochemistry, as described below.
  • Sections were then washed again in PBS (3x 10 min, at RT) and incubated in the secondary antibody (goat anti-rabbit Alexa 546 (Invitrogen), dilution 1 : 250 in PB containing 5% NGS, at RT for 3 hours). After washing in PB, the slices were stained for DAPI (dilution 1 :1000, at RT for 10 min) and mounted on glass slides using Vectashield medium (Vector Laboratories, Burlingame, CA, USA).
  • Imaging of phospho-Tau staining was performed on a confocal laser-scanning microscope (LSM 700, Carl Zeiss, Germany) using 10x and 63x objectives, and the confocal images were analyzed using Zen software (Carl Zeiss, Jena, Germany).
  • mice were first anesthetized with isoflurane in a chamber and were then placed into a stereotaxic apparatus on a 37 °C heating pad.
  • the levels of isoflurane and oxygen were set to 1.5 - 2% 0.4 l/min, respectively, and the breathing rate was used for monitoring the depth of anesthesia.
  • Ketoprofen (5 mg/kg) was injected to prevent inflammation and pain.
  • a lubricant ophthalmic ointment was applied. After cleaning the skin and shaving the fur, a cutaneous incision (length 8 mm) was made centrally over the frontal skull bone.
  • a cranial window with a diameter of 5 mm was drilled using a dental micro motor.
  • the center coordinates of the mPFC window were as follows: anterior-posterior (AP) +2.0 mm and medial-lateral (ML) 0 mm.
  • AP anterior-posterior
  • ML medial-lateral
  • the brain pial surface was cleaned with 0.9 % NaCI solution, and a 6.0-mm-diameter glass cranial window was implanted by applying a tiny layer of Roti Coll 1 superglue. The craniotomy was then sealed with dental cement.
  • animals were placed into a recovery chamber and injected with ketoprofen (5 mg/ kg) 24 h after the surgery.
  • mice were anesthetized intraperitoneally with ketamine (45 mg/kg body weight) and xylazine (18 mg/kg body weight) in 0.9 % NaCI solution and then placed in a head fixation frame, while body temperature was maintained at 37°C with a heating pad. Thirty minutes after injection, ⁇ 1 % isoflurane/0 2 gas mixture was employed to maintain anesthesia during in vivo microscopic imaging. Imaging was performed in the mPFC at the following stereotaxic coordinates: AP + 1.9 mm and ML ⁇ 0.5 mm.
  • mice After imaging of mice under basal conditions, they were removed from the head fixation frame to apply intranasally DMB-labelled DP2 or DP12 in the same manner as described for behavioral experiments, but at concentration of 10 mg/kg, to increase the fluorescent signal. The imaging session was then continued for up to 3 h and repeated 24 h after intranasal application.
  • DMEM modified Eagle’s medium
  • Example 1 Inhibition of GluN2B-EPSCs in prefrontal cortex slices by polySia fragments.
  • tegaserod a serotonin (5-HT 4 ) receptor agonist
  • 5-HT 4 a serotonin receptor agonist
  • tegaserod might potentially compete with polySia for binding to the polySia binding site of antibody 735 (Bushman et at, 2014).
  • the polySia-mimicking activity of tegaserod to stimulate peripheral nerve regeneration has been shown in vitro and in vivo and found to be independent of its described function as a 5-HT 4 receptor agonist (Bushman et at, 2014).
  • Example 2 Impaired LTP after endoNF treatment is restored by DP12.
  • TBS ta-burst stimulation
  • Example 3 Restoration of LTP in ST8SIA4-deficient mice by DP12.
  • mice that are constitutively deficient in the polysialyltransferase ST8SIA4 ( St8siaf /_ ) and that show no detectable polySia in the mPFC during adulthood (Eckhardt et al., 2000; Nacher et al., 2010).
  • Extracellular fEPSP recordings demonstrated a pronounced decrease in LTP magnitude (by ⁇ 40 %) in SfSs/a ⁇ - mice compared with St8sia4 +/+ mice ( Figure 4(A, E)), which resembled the LTP deficits found in endoNF-treated slices ( Figure 3(A, D)). This result could be reproduced in an independent set of recordings on another setup and by another set of LTP experiments performed at 35 °C (data not shown).
  • Example 4 Impaired object recognition memory in polySia-depleted mice is normalized by DP12.
  • the prefrontal cortex is known to be involved in object recognition memory in rodents (Barbosa et al., 2013). Because LTP was impaired in mPFC slices treated with endoNF, we asked whether acute removal of polySia in the prefrontal cortex would affect this form of learning and memory. Thus, we performed a longitudinal experiment comparing performance of C57BL/6J mice before and after endoNF injection into the mPFC in vivo, which efficiently digested polySia (Figure 5(A)). Two days before administration of endoNF, mice showed normal object recognition being i.p. injected with either vehicle (d-2) or sarcosine (d-1 ) 30 min before the encoding phase ( Figure 5(B)).
  • Example 5 Delivery of DP12 to mPFC by intranasal administration.
  • Example 6 Impaired object recognition memory in polySia-deficient mice is normalized by DP12.
  • St8sia4 +/+ mice showed a clear preference for the least as compared to the most recently explored object in the retrieval phase.
  • SfSs/a ⁇ - mice explored the two objects equally ( Figure 5(D)). Their performance in this task could be restored by DMB-DP12, but not by DMB-DP2.
  • Example 7 Impaired mPFC LTP and recent object recognition memory in mice overexpressing GFP-Tau[R406W] are normalized by DP12.
  • AAV-GFP-Tau[R406W] infected mice had impaired mPFC LTP, but bath application of DP12 could fully rescue LTP (Figure 7(C)): The levels of LTP after this treatment were not different from those in control AAV-GFP expressing mice. Consistent with data shown in Figure 5, control AAV-GFP-injected mice showed discrimination between recent (R) and less recent (L) objects. Untreated AAV-GFP-Tau[R406W] injected mice showed impaired recent object recognition, which was fully rescued by intranasal delivery of DP12, but not by control DP1 ( Figure 7(D)).
  • Example 8 Increase of LTP in CA3-CA1 synapses in slices from 5xFAD mice after treatment with DP12.
  • Example 9 Impaired recent object recognition memory in 5xFAD mice is normalized by DP12.
  • Example 10 DP12 and avDP10-9OAc do not affect the cell viability, while DP10 improves it.
  • Example 11 Impaired recent object recognition memory in 5xFAD mice is normalized by DP10 but not DP20.
  • the invention is further characterized by the following items:
  • Neu5Ac is N-acetylneuraminic acid
  • n is an integer in the range from 6 to 13
  • derivatives for use in the prevention or treatment of a neurological and neuropsychiatric disorder, wherein said derivatives are substituted with at least one sugar, acetylgroup, or acylgroup at at least one monomer of the polysialic acid,
  • n is an integer in the range from 10 to 13.
  • polysialic acid of item 1 or 2 wherein the polysialic acid inhibits the activation of heterodimeric GluN1/GluN2B or heterotrimeric GluN1/GluN2A/GluN2B-containing NMDA receptors.
  • polysialic acid of any one of the preceding items wherein said derivatives are substituted with at least one sugar, acetylgroup or acylgroup at the one monomer at the non- reducing end of the polysialic acid.
  • polysialic acid of any one of the preceding items wherein the at least one sugar is glucose, N-acetylglucosamine, N-acetylgalactosamine, galactose, fucose, mannose or xylose.
  • R is a carboxyl group
  • each of R 2 , R 3 , R 5 , R 7 , R 8 , R 10 , R 12 ⁇ R 13 is independently from each other a hydrogen; iii) R 4 is a hydroxyl group or an O-acetylgroup;
  • R 6 is NHCOCH 3 ;
  • R 9 is a hydroxyl group or an O-acetylgroup
  • Rn is a hydroxyl group or hydrogen
  • R 14 is a hydroxyl group or an O-acetylgroup.
  • R 4 is a hydroxyl group
  • R 6 is NHCOCH 3
  • R g is a hydroxyl group or an O-acetylgroup
  • R 14 is an O-acetylgroup.
  • n is an integer in the range from 10 to 13.
  • the neurological and neuropsychiatric disorder is schizophrenia, tauopathy, bipolar disorder, depression, epilepsy, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis or stroke.
  • tauopathy comprises Alzheimer’s disease, frontotemporal dementia (FTD), primary age-related tauopathy (PART), chronic traumatic encephalopathy, progressive supranuclear palsy (PSP), corticobasal degeneration, dementia with Lewy Bodies, frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP- 17), argyrophilic grain disease (AGD), glial globular tauopathy, Pick’s diseases, Parkinson’s disease.
  • FTD frontotemporal dementia
  • PART primary age-related tauopathy
  • PSP progressive supranuclear palsy
  • corticobasal degeneration dementia with Lewy Bodies
  • frontotemporal dementia and parkinsonism linked to chromosome 17 FTDP- 17
  • ATD argyrophilic grain disease
  • glial globular tauopathy Pick’s diseases, Parkinson’s disease.
  • a pharmaceutical composition comprising as an active ingredient the polysialic acid and/or derivatives and/or pharmaceutically acceptable salts thereof according to any one of the preceding items, optionally comprising a pharmaceutical acceptable carrier.
  • the pharmaceutically composition comprises a pharmaceutically acceptable carrier, wherein the pharmaceutical acceptable carrier is a solid pharmaceutical acceptable carrier, preferably lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate or stearic acid, a liquid pharmaceutical acceptable carrier, preferably sugar syrup, peanut oil, olive oil or water, or a gaseous pharmaceutical acceptable carrier, preferably carbon dioxide or nitrogen.
  • the pharmaceutical acceptable carrier is a solid pharmaceutical acceptable carrier, preferably lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate or stearic acid
  • a liquid pharmaceutical acceptable carrier preferably sugar syrup, peanut oil, olive oil or water
  • a gaseous pharmaceutical acceptable carrier preferably carbon dioxide or nitrogen.
  • parenteral administration comprises intravitreal injection, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, perfusion, infusion or topical administration.
  • the pharmaceutical composition of item 17, wherein the neurological and neuropsychiatric disorder is schizophrenia, tauopathy, bipolar disorder, depression, epilepsy, Huntington’s disease, multiple sclerosis, amyotrophic lateral sclerosis, or stroke.
  • tauopathy comprises Alzheimer’s disease, frontotemporal dementia (FTD), primary age-related tauopathy (PART), chronic traumatic encephalopathy, progressive supranuclear palsy (PSP), corticobasal degeneration, frontotemporal dementia, dementia with Lewy Bodies, frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), argyrophilic grain disease (AGD), glial globular tauopathy, Pick’s diseases, and Parkinson’s disease.
  • step b) stopping the reaction of step a) with NaOH;
  • Tegaserod mimics the neurostimulatory glycan polysialic acid and promotes nervous system repair. Neuropharmacology 79, 456-466.
  • Glycine tranporter-1 blockade potentiates NMDA-mediated responses in rat prefrontal cortical neurons in vitro and in vivo. Journal of neurophysiology 89, 691-703.
  • Neural cell adhesion molecule-associated polysialic acid inhibits NR2B- containing N-methyl-D-aspartate receptors and prevents glutamate-induced cell death. J Biol Chem 281, 34859-34869.

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Abstract

La présente invention concerne un acide polysialique répondant à la formule générale (1) comme suit et ses dérivés : (α(2 → 8)Neu5Ac)n, n étant un nombre entier situé dans la plage allant de 6 à 13, destiné à être utilisé dans la prévention ou le traitement de troubles neurologiques et neuropsychiatriques. La présente invention concerne également une composition pharmaceutique comprenant, en tant que principe actif, ledit acide polysialique et/ou des dérivés associés et/ou des sels pharmaceutiquement acceptables associés. En outre, la présente invention concerne un procédé de production dudit acide polysialique et/ou de sels pharmaceutiquement acceptables associés.
PCT/EP2019/070576 2018-07-31 2019-07-31 Acide polysialique et ses dérivés, composition pharmaceutique et procédé de production d'acide polysialique WO2020025653A2 (fr)

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